T Cell Activation with anti-CD3 Antibodies 

 Human T Cell Activation with anti-CD3
(clone UCHT1 or HIT3a)
Materials:
• Sterile PBS
• Anti-human CD3, clone UCHT1 (LEAF™ format, Cat. No. 300413/4) or clone
HIT3a (LEAF™ format, Cat. No. 300313/4)
• Cell culture medium (e.g., RPMI-1640 or IMDM supplemented with 10% FBS
and 2mM L-glutamine)
• Sterile single-cell suspension of Ficoll-Hypaque-purified peripheral blood
mononuclear cells, isolated T cells, or T cell subsets
• 96-well flat-bottom tissue culture plates with lids (e.g., Costar? Cat. No. 3596)
Method:
1. Prepare a 10 μg/ml solution of anti-CD3 (clone UCHT1 or HIT3a) in sterile
PBS.
2. Dispense 50 μl of the antibody solution to each microwell of the 96-well assay
plate. For the unstimulated control wells, add 50 μl of sterile PBS.
3. Seal plate. Incubate at 37°C for 2 hours or 4°C overnight.
4. Aseptically decant antibody solution from the microwell plate.
5. Wash plate microwells 3 times with sterile PBS. Discard liquid.
6. Prepare single cell suspension of cells of interest in supplemented cell culture
medium to 1-2 x 106/ml.
7. Aliquot 200 μl cell suspension into plate microwells. Cover with lid. Incubate
at 37°C in 5% CO2 and 100% humidity for 3 days.
* Soluble forms of LEAF™ purified UCHT1 (1 μg/ml) or LEAF™ purified HIT3a
(0.01 – 0.1 μg/ml) may be used to activate T cells from PBMC cell populations.

biolegend.com
Rev: 030107
Mouse T Cell Activation with anti-CD3ε
(clone 145-2C11)
Materials:
• Sterile PBS
• Anti-mouse CD3ε, clone 145-2C11 (LEAF™ format, Cat. No. 100313/4)
• Cell culture medium (e.g., RPMI-1640 or IMDM supplemented with 10% FBS
and 2mM L-glutamine)
• Sterile, single-cell suspension (e.g., splenocytes, lymph node cells), isolated
T cells or T cell subsets
• 96-well flat-bottom tissue culture plates with lids (e.g., Costar? Cat. No. 3596)
Method:
1. Prepare a 5 μg/ml solution of anti-CD3ε (clone 145-2C11) in sterile PBS.
2. Dispense 50 μl of the antibody solution to each microwell of the 96-well assay
plate. For the unstimulated control wells, add 50 μl of sterile PBS.
3. Seal plate. Incubate at 37°C for 2 hours or 4°C overnight.
4. Aseptically decant antibody solution from microwell plate.
5. Wash plate microwells 3 times with sterile PBS. Discard liquid.
6. Prepare single cell suspension of cells of interest.
7. Resuspend cells in supplemented cell culture medium to 1-2 x 106/ml.
8. Aliquot 200 μl cell suspension into plate microwells. Cover with lid. Incubate
at 37°C in 5% CO2 and 100% humidity for 3 days.

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